Stabilized occult blood diagnostic



United States Patent 3,252,762 STABILIZED OCCULT BLOOD DIAGNOSTIC Ernest C. Adams, Jr., and Norman R. Novak, Elkhart, Ind., assignors to Miles Laboratories, Inc Elkhart, Ind., a corporation of Indiana No Drawing. Filed May 4, 1961, Ser. No. 107,646 20 Claims. (Cl. 23-253) This invention relates to a composition which has utility in the detection of blood. Particularly, the invention relates to compositions which are suitable for use in the qualitative detection and quantitative estimation of blood in body fluids such as urine, vomitus, gastric content, cerebral spinal fluids and in feces. More particularly, the invention relates to blood testing compositions wherein one component of the test is encapsulated by means of a novel encapsulating material.

The detection of occult blood in body fluids and feces has become an invaluable aid to the medical practitioner in the diagnosis of a great number of disorders. Blood is found in the gastric contents and in vomitus in conditions associated with erosion of the mucous membranes in ulcers and incarcinomas. In the feces the regular and frequent occurrence of occult blood is suggestive of gastro-intestinal cancer, gastric or duodenal ulcers or hemorrhoids. In these conditions the hemorrhage is often so slight that it is not possible to detect blood by microscopic identification of the erythrocytes (red blood cells) and a sensitive and specific chemical test for occult blood becomes invaluable. In the urine blood cells (hematuria) or blood pigment (hemoglobinuria) is found in typhus, scurvy, purpura, pyemia, nephritis, renal calculi, as the result of a burn covering a large part of the body, as a result of the action of various hemolytic toxins and the like.

The prior art has recognized the need for a simple, reliable test for occult blood. US. Patent No. 2,290,436, issued July 21, 1942, to Kamlet, U.S. Patent No. 2,799,- 660, issued July 16, 1957, to Nicholls and Former and US. Patent No. 2,838,377, issued June 10, 1958, to Fonner (all assigned to the instant assignee) illustrate various test compositions which have been supplied to meet this need.

The instant inventive concept, like those of the prior art, is based on the catalytic activity of the prosthetic groups present in blood. These catalytically active substances, identified in hemoglobin, belong to the general class of hemoproteins, conjugate proteins all of which have the same prosthetic groups, iron protoporphyrin or heme. This prosthetic group has the ability to catalyze the transfer of oxygen from an oxygen source to an acceptor which in turn becomes oxidized. If the acceptor is a dye precursor, colorless until it becomes oxidized and colored in its oxidized form, then the presence of the catalytic activity is indicated by color formation.

Thus, the composition of this invention comprises an indicator or dye precursor plus an oxygen source. In the presence of hemoglobin of blood which contains the catalytic prosthetic group, the transfor of oxygen from the oxygen source to the acceptor or dye precursor will occur and the indicator will become oxidized and colored. The presence of color, then, is an indication of the presence of blood. The rapidity of the color change and the depth or density of the color, when compared to a set'of standards, is a means of the quantitative estimation of the blood present.

The instant invention is directed to an improvement over the prior art techniques of testing for blood, particularly occult blood in body fluids and in'other substances. It has now been found that an improved test composition results when one of a group of organic hydroperoxides having pronounced sensitivity in the indicator reaction above described is encapsulated or entrapped within a colloidal material such as gelatin, which has been hardened by fixing with a dialdehyde polysaccharide which is efiective for this purpose, and combined in this form along with a suitable indicator and buffering means.

As pointed out above, a certain group of organic hydroperoxides has been found to have the pronounced sensitivity which is required for the indicator reaction to be utilized in the diagnostic compositions of this invention. The hydroperoxides consist of a group of materials including cumene hydroperoxide, diisopropylbenzene hydroperoxide, paramenthane hydroperoxide, and 2,5-dimethylhexane 2,5-dihydroperoxide to mention the most significant members of this group. Other organic hydroperoxides of similar structure may likewise be used if desired.

While the use of the above described organic hydroperoxides has been found to produce outstanding results in various occult blood diagnostic compositions, these hydroperoxides when mixed with various other materials which are required in the provision of diagnostic compositions of this nature are found to be less stable than might be desired. Accordingly, there is provided in this invention a novel encapsulating material for the organic hydroperoxides. This encapsulating material may be comprised of various protein or polysaccharide materials such as gelatin, algin, carrageenin, casein, albumin or other materials of this nature. These various protein or polysaccharide materials are applied to the test compositions and then hardened by means of a fixing process, which involves treating with a dialdehyde polysaccharide which is effective for this purpose.

In general it has been found desirable to mix the organic hydroperoxide being used with an emulsifying agent such as acacia mucilage to form a primary emulsion. Other emulsifying agents which may be used include polyvinyl alcohol, gum arabic, carboxy vinyl polymer and the like. If desired, a surfactant or wetting agent such as dioctyl sodium sulfosuccinate may also be used. The emulsifying agent serves to lower the surface tension of the oily organic hydroperoxide and forms a film around each individual oil droplet. The surfactant, if used, assists in this regard. The resulting primary emulsion is buffered by means of an appropriate buffer such as a mixture of sodium citrate and citric acid. Preferably, the butter is first mixed with the material which is to form the encapsulation for the organic hydroperoxide such as gelatin before addition to the hydroperoxide emulsion.- Various materials may be used for the encapsulation as pointed out above. An aqueous dispersion of a dialdehyde polysaccharide, such as dialdehyde starch, is next added to serve as a fixing agent for the encapsulating material. The encapsulation material which is present in the film around the oily organic hydroperoxide droplets is thereby fixed by the dialdehyde polysaccharide resulting in a more stable preparation.

It should be noted that thorough mixing is of extreme importance at each addition. This is particularly so upon the addition of the buffer-encapsulation mixture to the primary emulsion ofthe organic hydroperoxide. The addition of a suitable surfactant or wetting agent such as sodium lauryl sulfate has been found to enhance mixing of the various materials and to result in a composition which is not only sensitive but stable as well.

This composition is then buffered and may be applied O hydroperoxide and is the preferred buffer for this purpose, there may be used any other suitable buffer material, such as, for example, a tartrate, phosphate, phthalate, acetate or other buffer. The preferred range of hydrogen ion concentration to which the composition is buffered is from about pH 4 to pH 7.

As pointed out above, the encapsulating material to be used in the diagnostic compositions of this invention is fixed or hardened by treatment with a dialdehyde polysaccharide. Dialdehyde polysaccharides, of which dialdehyde starch is an example, may be prepared by the well known oxidation of polysaccharides with periodic acid. This preparation is illustrated by the conversion of starch to dialdehyde starch using periodic acid as the oxidizing agent in accordance with the following equation CIHQOH O H H jg H104 011 H L |i H OH x TIIiOH -O H H H O O x wherein x stands for the number of repeating units in the molecule, which may range from to several thousand.

The dialdehyde polysaccharides to be used in this process may be the dialdehyde derivatives'of any polysaccharide such as corn, wheat, tapioca or potato starches, celluloses, dextrans, algins, inulin or others. Of these polysaccharides, the dialdehyde derivatives of starch, known generically as dialdehyde starch, are the best known and most often used. However, where it is desired to have derivatives of other polysaccharides, these may also be used.

In general, it is preferred to use dialdehyde polysaccharides which are from about 50% to 100% oxidized, that is, those wherein 50 to 100 of the original anhydroglucose units have been converted to dialdehyde units, such as by periodate oxidation.

In this invention, the dialdehyde polysaccharide is used in the form of an aqueous dispersion, which may be readily prepared by adding the dialdehyde polysaccharide in the desired concentration, usually from about 0.25% to about 1.5%, to tap water or to a buffer solution and then mixing the aqueous dispersion until a relatively homogeneous dispersion is obtained. The dialdehyde polvsaccharide may be used in whatever concentration is required to satisfactorily harden the encapsuating material to provide the desired stability features of the instant invention. The precise concentration to be used will be pointed out more particularly hereafter.

Although orthotolidine is the preferred indicator for the diagnostic compositions of this invention, various other indicator materials may be used so long as they satisfy the requirements pointed out above, namely that they undergo a color change in the presence of an oxygen source and the blood for which the unknown material is being tested. Such indicators comprise a variety of organic materials, principally those of the aniline and phenol derivatives. To name but a few, there may be used in addition to orthotolidine, o-toluidine, p-toluidine, o-phenylenediamine, N,N-dimethylp-phenylenediamine, N,N-diethyl-p-phenylenediamine, benzidine, p-anisidine, di-anisidine, o-cresol, m-cresol, p-cresol, a-naphthol, 8- naphthol, catechol, guaiacol and pyrogallol.

While the novel compositions of this invention may be incorporated in any of various diagnostic media, it has been found that incorporating these compositions upon discs or strips of paper produces the most useful and most satisfactory diagnostics. In particular, sticks of bibulous material, such as rigid paper sticks, have been found to be eminently satisfactory for this purpose.

Although the range of compositions of the diagnostic materials provided by this invention may vary within fairly wide limits in accordance with the general teachings set forth above, it has been found that particularly useful compositions can be provided by using the various ingredients within the ranges set forth in the table below, which is an illustration of the types of compositions which may be used in this invention rather than a limitation of the total entire useful range to the precise concentrations set forth.

TABLE Emulsifying agent (60% w./-v.), ml -100 Organic hydroperoxide, ml. 2.5-10 Surfactant for hydroperoxide emulsion, ml. 0-2 Buffer I, ml. 25-50 Encapsulating material, g 0.7-3.5 Dialdehyde polysaccharide (1% w./v.), ml. 0.4-3

Surfactant for other ingredients (5% w./v.),

ml. 3.75-12 Buffer II (3 X concentration of Buffer 1), ml. 25-75 Example I To a mortar there was added 100 ml. of acacia mucilage (made up by adding 200 g. of acacia to 500 ml. of boiling water), followed by 2 ml. of dioctyl sodium sulfosuccinate (5 w./v.). There was then added 5 ml. of cumene hydroperoxide and the mixture was triturated for 5 minutes to form a primary emulsion. Next, a citrate buffer solution was made up by heating 1,500 ml. of water to boiling and adding it to a mixture of 163 g. of sodium citrate and 37 g. of citric acid. The citrate buifer was then mixed with 0.7 g. of gelatin and 50 ml. of the buffergelatin mixture was added to the primary emulsion and mixed well. Next was added 1 ml. of a 1% dispersion of dialdehyde starch with thorough mixing. A 5% solution of sodium lauryl sulfate was then added to the mixture in an amount of 12 ml. The resulting composition was then homogenized by means of a hand homogenizer and 50 ml. of a buifer solution prepared as above using 500 ml. of water, 163 g. of sodium citrate and 37 g. of citric acid was added with mixing. The finished emulsion was then applied to paper strips by dipping and the impregnated paper strips were then dried in an oven at C. to C. for 24 hours. Finally, 320 mg. of orthotolidine was dissolved in 16 ml. of choloroform and applied to the dried paper strips by dipping. The excess chloroform was evaporated from the strips by means of mild heat. These strips were then tested on various urine samples and showed positive on samples containing one part blood in 30,000 parts urine.

To demonstrate the increased stability of the diagnostic stick prepared according to the teachings of this invention, a comparison was made between sticks in which no fixative was used, some in which formaldehyde was used as the fixative and some prepared according to this invention using a dialdehyde polysaccharide as the fixative. The sticks thus prepared were tested under various conditions of temperature and relative humidity, in each case for a seven day period. The comparative results of these tests are shown in Table 1 wherein A refers to the sticks in which no fixative was used, B refers to those in which formaldehyde was used as a fixative and C refers to those prepared according to this invention, that is, those in which a dialdehyde polysaccharide was used as the fixative.

TABLE 1 Conditions Results Relative Temperature humidity,

percent 40 C 70 All discolored, no difference. 40 C 50 C'least discolored and best sensitivity;

, B- worst. 40 C 35 C and A were less discolored and more sensitive than B. 70 C All some discoloration; C-most sensitive. 60 C Crnost sensitive. 50 C C-best; B-worst. 40 Do. Room Do.

Example II To a 1,000 ml. lbeaker was added 500 ml. of acacia mucilage, prepared by adding 500 ml. of boiling water to 200 g. of acacia, and 25 ml. of cumene hydroperoxide. These ingredients were mixed with a Lightning mixer for minutes at a rheostat setting of 70-80. In another beaker 1,500 ml. of boiling water was added to a mixture of 163 g. of sodium citrate and 37 g. of citric acid. Upon cooling 3.5 g. of gelatin was dissolved in the citrate buffer and the mixture was added to the beaker containing the acacia mucilage and cumene hydroperoxide. Next there was added 5 ml. of a 1% w./v. dispersion of dialdehyde starch. The dialdehyde. starch was allowed to mix with the'other ingredients and then 50 ml. of sodium lauryl sulfate was added and mixing continued for 5 minutes at a rheostat setting of 60. The resulting emulsion was passed through a Manton-Gaul-in homogenizer for 5 minutes at 2,000 lb. pressure. To the homogenized mixture was added 250 ml. of a citrate buifer which had previously been prepared by adding 500 m1. of boiling water to a mixture of 163 g. of sodium citrate and 37 g. of citric acid and mixing continued. The emulsion was then applied to paper strips by dipping and the impregnated strips were placed in a kiln at 160 F. for 16 hours. Finally, the strips were dipped into a chloroform solution of orthotolidine which had been prepared by dissolving 1.28 g. of orthotolidinc in 64 ml. of chloroform and the excess chloroform allowed to evaporate by use of the kiln. These strips were then tested in various urine samples and were shown to be sensitive to one part of blood in 25,000 parts of urine.

In summary, vthe invention relates to novel diagnostic compositions for the detection of occult blood in various fluids, which include an organic hydroperoxide encapsulated in a polysaccharide or protein material which has been hardened or fixed by treatment with dialdehyde polysaccharide buffered at a pH of from pH 4 to pH 7 and an indicator material which is capable of undergoing a color change by virtue of oxygen transfer between the aforementioned organic hydroperoxide and the indicator in the presence of blood. These compositions are particularly useful for incorporation in diagnostic discs or sticks.

What is claimed is:

1. A test composition in dry solid form for the detection of blood which comprises an encapsulated organic hydroperoxide selected from the group consisting of cumene hydroperoxide, diisopropylbenzene hydroperoxide, paramenthane hydroperoxide, and 2,5-dimethy1hexane 2,5-dihydroperoxide, an indicator capable of accepting a catalytic transfer of oxygen from the hydroperoxide by means of the prosthetic group of hemoglobin and be coming oxidized with an accompanying color change, a buffer maintaining the pH of material being tested to one within the range of about from pH 4 to pH 7, said organic hydroperoxide being encapsulated by means of a dialdehyde polysaccharide fixed gelatin.

presence of said organic hydroperoxide and blood, said organic hydroperoxide being encapsulated upon said bibulous carrier by means of a dialdehyde polysaccharide fixed gelatin.

6. A device according to claim 5 wherein the dialdehyde polysaccharide is dialdehyde starch.

7. A device according to claim 5 wherein the organic hydroperoxide is cumene hydroperoxide.

8. A device according to claim 5 wherein the indicator material is orthotolidine.

9. A test composition in dry solid form for the detection of blood, which comprises an encapsulated organic hydroperoxide selected from the group consisting of cumene hydroperoxide, diisopropyI-benzene hydroperoxide, para-menthane hydroperoxide and 2,5-dimethyl hexane 2,5-dihydroperoxide, an indicator capable of accepting a catalytic transfer of oxygen from the hydroperoxide by means of the phosthetic group of hemoglobin and becoming oxidized with an accompanying color change, a buifer maintaining the pH of the test composition within the range of about from pH 4 to pH 7, said organic hydroperoxide being encapsulated by means of a dialdehyde polysaccharide fixed gelatin and as a surfactant, sodium lauryl sulfate.

10. A composition according to claim 9 wherein the dialdehyde polysaccharide is dialdehyde starch.

11. A composition according to claim 9 wherein the organic hydroperoxide is cumene hydroperoxide.

12. A composition according to claim 9 wherein the indicator material is orthotolidine.

13. A device in dry solid form for the detection of blood, which comprises a bibulous carrier and incorporated therewith, an encapsulated cumene hydroperoxide and orthotolidine, said cumene hydroperoxide being encapsulated by means of a dialdehyde polysaccharide fixed gelatin.

14. A process for the preparation of a test composition for the detection of blood which comprises mixing an organic hydroperoxide selected from the group consisting of cumene hydroperoxide, diiso propylbenzene hydroperoxide, paramenthane hydroperoxide and 2,5-dimethylhexane 2,5-dihydroperoxide with an emulsifying agentto form a primary emulsion,

mixing the primary emulsion with an admixture of a pH 4 to pH 7 buffer, a protein and a surfactant to form an encapsulated material,

fixing the protein with a dialdehyde polysaccharide,

and

incorporating the fixed encapsulated material with an indicator capable of accepting a catalytic transfer of oxygen from the hydroperoxide by means of the prosthetic group of hemoglobinand becoming oxidized with an accompanying color change.

15. A process according to claim 14 wherein the emulsifying agent is gum arabic.

16. A process according to claim 14 wherein the organic hydroperoxide is cumene hydroperoxide.

17. A process according to claim 14 wherein the surfactant is sodium lauryl sulfate.

- 7 18. A process according to claim 14 wherein the indicator is o-tolidine.

19. A process according to claim 14 wherein the protein is gelatin.

20. A process according to claim 4 wherein the dialdehyde polysaccharide is dialdehyde starch.

References Cited by the Examiner UNITED STATES PATENTS 2,290,436 7/ 1942 Kamlet. 2,754,289 7/1956 Meyer. 2,799,660 7/ 1957 Nicholls et a1. 2,800,458 7/1957 Green. 2,886,445 5/ 1959 Rosenthal. 2,953,454 9/ 1960 Berman.

8 Collins. Fancher. Jensen. Jeffreys et a1. 96111 XR Adams et a1.

MORRIS O. WO LK, Primary Examiner.

GEORGE D. MITCHELL, Examiner.

15 O. F. CRUTCHFIELD, Assistant Examiner. 

5. A TEST DEVICE IN DRY SOLID FORM FOR THE DETECTION OF BLOOD WHICH COMPRISES A BIBULOUS CARRIR AND INCORPORATED THEREWITH, AN ENCAPSULATED ORGANIC HYDROPEROXIDE SELECTED FROM THE GROUP CONSISTING OF CUMENE HYDROPEROXIDE, SIISOPROPYLBENZENE HYDROPEROXIDE, PARA-MENTHANE HYDROPEROXIDE, AND 2,5-DIMETHYLHEXANE 2,5-DIHYDROPEROXIDE, AND AN INDICATOR MATERIAL WHICH CHANGES COLOR IN THE PRESENCE OF SAID ORGANIC HYDROPEROXIDE AND BLOOD, SAID ORGANIC HYDROPEROXIDE BEING ENCAPSULATED UPON SAID BIBULOUS CARRIER BY MEANS OF A DIALDEHYDE POLYSACCHARIDE FIXED GELATIN. 